Identification of small molecules that interfere with H1N1 influenza A viral replication

Abstract

Successful replication of the influenza A virus requires both viral proteins and host cellular factors. In this study we used a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We used an established reporter assay to assess influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, which inhibits a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose-dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 is effective in rescuing high-dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a new and potentially suitable scaffold for the development of anti-influenza agents that act on novel targets.

Figure 1. Diagrams of the wild type A/WSN/33 virus and the modified WSN-Ren luciferase virus, diagram of WSN-Ren assay and dose response curves of compounds 7 and 8

a) Representative diagrams show the genomic makeup and surface viral proteins of a wild type WSN influenza virus and of a WSN-Ren luciferase virus. The modification is engineered by replacement of viral hemagglutinin (HA) coding sequence with that of Renilla luciferase. b) A diagram demonstrates WSN-Ren luciferase assay and c) Dose dependent inhibitory activity of compound 7 and compound 8 in the WSN-Ren luciferase assay with EC₅₀ values of 45.6 nM and 86.7 nM respectively.

Figure 2. Inhibition of viral replication by compound 7

a) Influenza NP of WSN strain (WSN-NP) mRNA expression level in MDCK-HA cells after WSN-Ren luciferase virus infection. Cells were treated with indicated concentrations of compound 7 or DMSO during infection. Cell lysates were collected at 3, 7, 11, 14 and 26 hour for total mRNA preparation. The WSN-NP mRNA level was then measured in quantitative real time PCR (qRT-PCR) and standardized against the mRNA level of β-actin. b) The WSN-NP mRNA expression level in A549 cells after wild type influenza A/WSN/33 virus infection. A549 cells were treated with indicated concentrations of compound 7 or DMSO during infection. Cell lysates were collected at 3.5, 7, 11, 23, 27 and 31 hour for total mRNA preparation. The WSN-NP mRNA level was then measured in qRT-PCR and standardized against the mRNA level of human TATA box binding protein.

Figure 3. Mouse survival curve

Mice were infected with high dose mouse-adapted PR/8 influenza virus at day 0. Mice were treated every 12 hour via i.p. route for 5 days then monitored for survival. Survival curves demonstrated that 50% of compound 7 (100 mg/kg/day) treated mice, 80% of mice treated with oseltamivir phosphate (1 mg/kg/day) and 30% of the mice treated with PBS survived.

Scheme 1. Synthesis of 3-(((2-hydroxyethyl)(2-methyl-1-(1-(tert-pentyl)-1H-tetrazole-5-yl)propyl)amino)methyl)-6,7-dimethylquinolin-2(1H)-one (Compound 7)

Reagents and conditions: (a) Ac₂O, Et₃N, CH₂Cl₂, R.T., 1 h; (b) POCl₃, DMF, 90 oC, 16 h; (c) AcOH, H₂O, reflux, 5 h; (d) 2-aminoethanol, AcOH, 80 oC, 3 h; (e) NaBH₄, EtOH, R.T., 2 h; (f) TMSN₃, isobutyraldehyde, t-amylisocyanide, 50 oC, 2 h.

Scheme 2. Synthesis of compound 23

Reagents and conditions: (a) TMSN₃, isobutyraldehyde, t-amylisocyanide, R.T.

Scheme 3. Synthesis of compound 24

Reagents and conditions: (a) benzoic acid, EDC, HOBt, DIEA, DMF, 70 °C, 4 h.

Scheme 4. Synthesis of (Compound 25)

Reagents and conditions: (a) (tetrahydrofuran-2yl)methanamine, AcOH, 80 °C, 3 h then NaBH₄, EtOH, R.T., 2 h; (b) TMSN3, isobutyraldehyde, t-amylisocyanide, 50 °C, 2 h.